1. Field of the Invention
This invention relates to a process for separating isoenzymes from tissue extracts or blood sera using ion exchange chromatography. More specifically, this invention relates to a process for separating and isolating from tissue extracts or blood sera clinically significant isoenzymes, the level of which is useful in diagnosing hepatic, myocardial and prostatic disorders.
2. Description of the Prior Art
In disease of organs, various items specific for the tissue involved are emitted into the bloodstream. As a result, it has become routine diagnostic procedure to sample the blood to obtain a general profile of the health of many parts of the anatomy as opposed to doing exploratory surgery.
More specifically, it is well documented that the level of various enzymes is elevated in certain disease conditions. In particular, an increase in the level of lactic acid dehydrogenase (EC 1.1.1.27, hereinafter LDH for brevity) in blood serum occurs with myocardial infarction, liver disease; pulmonary disease, etc. Further, the level of the enzyme creatine phosphokinase (EC 2.7.3.2, hereinafter CPK for brevity) is elevated in the blood serum in a similar manner when myocardial infarction, as well as skeletal muscle trauma, muscular dystrophy, and other disorders occur. Also, an increase in the level of the enzyme acid phosphatase (EC 3.1.3.2, orthophosphoric monoester of phosphohydrolase (acid optimum), hereinafter AP for brevity) occurs due to disorders of the recticulo-endothelial system, of the blood and of the prostate gland.
In general, enzymes are present in multiple forms, with various forms being specific for certain organs. These multiple forms of the enzymes, generally called "isoenzymes", have the same function in each organ, have the same average molecular weight but differ in their molecular charges or differ in the degree of electrical charge. In the past, the identification of various isoenzymes has been accomplished by separating the various isoenzymes using electrophoresis on a supporting medium, such as a paper or gel medium.
With respect to LDH, clinically useful in diagnosing anatomical disorders, it has been found by electrophoretic analysis to exist in five multiple forms in blood sera. (See E. S. Vesell et al, Proc. Soc. Exp. Biol. Med., 94, 96 (1957); T. Wieland et al, Biochem. Z., 329, 112 (1957); F. Wroblewski et al, Ann. N.Y. Acad. Sci., 94, 912 (1961); R. Richterich et al, Clin. Chim. Acta, 8, 178 (1963); and E. D. Wachsmuth et al, Biochem. Z., 336, 545 (1963)). These five multiple forms are generally designated LDH.sub.1, LDH.sub.2, LDH.sub.3, LDH.sub.4 and LDH.sub.5, with LDH.sub.1 being the form which is most highly negatively charged and LDH.sub.5 being the form which is least highly negatively charged, with the other forms varying in order in the degree of their electrical charge. By testing tissue extracts and blood sera and correlation of not only the level of but also the form of LDH, it has been found that LDH is specific for heart muscle and the level of LDH.sub.1, and also of LDH.sub.2, in blood sera is elevated in myocardial infarction as disclosed in C. R. Roe et al, J. Lab. Clin. Med., 80, 577 (1972). In a similar manner, LDH.sub.5 has been found to be specific for the liver and the level of LDH.sub.5 is elevated in blood sera in hepatic disorders. (See R. J. Wieme et al, Ann. N.Y. Acad. Sci., 94, 898 (1961); L. Cohen et al, Med. Clin. N. Am., 50, 193 (1966); I. N. Ramdeo et al, Am.J. Gastroenterol, 55, 459 (1971); B. E. Sobel et al, Circulation, 45, 471 (1972)).
Electrophoretic analysis has also shown that CPK is present in three forms, CPK-MM, CPK-MB, and CPK-BB (as disclosed in D. M. Dawson et al, Biochem. Biophys. Res. Comm., 21, 346 (1965)), with these three forms increasing in their negative charge respectively. It has been found that CPK-MM is found in skeletal muscle, heart and lung, CPK-MB is found mostly in the heart and CPK-BB is found mostly in the brain and the gastrointestinal tract. (See K. J. Van Der Veen et al, Clin. Chim. Acta, 13, 312 (1966)). With respect to these CPK isoenzyme forms, studies have shown that the level of CPK-MM is elevated in subjects with severe muscle trauma, while subjects with myocardial infarction have elevated levels of both CPK-MM and CPK-MB. (See C. R. Roe et al, supra, and K. K. Van Der Veen et al, supra)
Further, the analysis of AP has also shown that AP exists in multiple forms and one form, prostatic acid phosphatase (hereinafter PAP for brevity), is specific to diseases of the prostate gland. (See W. H. Fishman et al, J. Biol. Chem., 200, 89 (1953), W. H. Fishman et al, J. Clin. Invest., 32, 1034 (1953), L. T. Yam, Am.J. Med., 56, 604 (1974) etc.).
In view of the diagnostic value, the importance of the isolation of and determination of the levels of various isoenzymes in tissue extracts and blood sera, for example, those described above, can be easily seen. Further, the necessity for substantially complete separation into the various forms for analysis is obviously quite important for diagnostic accuracy.
Accordingly, techniques whereby the various forms of isoenzymes, such as the isoenzymes described and discussed above, can be separated for meaningful diagnostic information are essential for use of such isoenzymes as diagnostic tools.
In the past, the classical procedure for identification of isoenzymes has been electrophoresis and in particular electrophoretic analysis and identification of CPK isoenzymes has been accomplished (see V. Anido et al, Am.J. Clin.Pathol., 63, 761 (1974)). Unfortunately, electrophoretic analysis to identify isoenzymes involves a tedious, cumbersome and time-consuming method. Further, degradation or denaturation of the components present in the tissue extract or blood serum sample can occur, giving rise to erroneous results, due to problems inherent in the electrophoretic procedure.
To overcome the problems inherent in the electrophoretic method, ion exchange chromatography has been employed as a method for separation and isolation of isoenzymes and the use of ion-exchange chromatography has been shown to be a rapid and simple procedure for separation and isolation of isoenzymes.
While not desiring to be bound, the process of ion exchange chromatography is believed to utilize the molecular charge of the isoenzymes to bind the isoenzymes reversibly to an ion-exchange material with selective elution and collection then being possible according to the changes in the environment to which the ion-exchange material having the isoenzymes bound thereto is subjected. More specifically, the isoenzymes of LDH, CPK and AP are negatively charged and will bind to a weakly basic anion-exchange resin. In general, when ion-exchange chromatography is employed, a sample of blood serum or tissue extract containing the isoenzymes initially present is added to a column packed with the ion-exchange material. As the sample passes through the ion-exchange material in the chromatographic column, the isoenzymes, due to their negative charge, become bound to the ion-exchange resin at the active sites of the resin with the degree of bonding being dependent upon the charge density. To recover the isoenzyme components present in a separate isoenzyme form, the isoenzymes bonded to the ion-exchange resin are eluted from the ion-exchange resin by changing the ionic strength and pH environment. A selective displacement of a certain isoenzyme or isoenzymes to the exclusion of others occurs. To recover the isoenzymes in a separated form, the ion-exchange resin is washed with different solutions of increasing ionic strength and decreasing pH, i.e., by washing the ion exchange resin successively with different salt solutions having increasing concentrations and decreasing pHs. Due to the successive increase in ionic strength a selective displacement of the isoenzymes in the order of their charge density occurs since their bonding strength to the ion-exchange resin is diminished due to the lower pH. As a result of this procedure, the most weakly negatively charged isoenzymes are eluted first and followed successively in order by an elution of or elutions of, depending upon the sequence of eluting solution additions, the more highly negatively charged isoenzymes which elute later. The change in the pH of the solution employed for elution gives rise to the ability to recover the more highly charged isoenzymes without the necessity to increase the ionic strength to a level at which the isoenzymes or the ion-exchange resin is destroyed.
Ion-exchange column chromatography has been employed in the past to separate both CPK and LDH isoenzymes into their individual isoenzyme forms. More specifically, K. Takahashi et al in Clin. Chim. Acta, 38, 285 (1972), describe a procedure whereby tissue extract and blood serum were subjected to a gradient elution in ion exchange chromatography using DEAE-Sephadex A-50 to obtain CPK isoenzymes. In this procedure, analysis was of 5 ml fractions using a total of 50 fractions to fractionate an initial 1 ml sample. The gradient elution in the ion exchange chromatography described was accomplished by using a salt gradient procedure employing a buffered sodium chloride solution of a concentration ranging from 0 to 0.5 M and a pH of 7.5. Unfortunately, the procedure described by Takahashi et al is tedious and time-consuming since such involves a gradient elution with multiple fractions being collected and analyzed for the presence of CPK isoenymes and, in fact, in many instances 50 different fractions were collected for analysis.
D. W. Mercer in Clin. Chem., 20, 36 (1974) and ibid, 20, 895 (1974) and subsequent investigators (see M. A. Varat et al, Circulation 51, 855 (1975) and D. A. Nealon et al, Clin. Chem., 21, 392 (1975)) disclose a technique involving step-wise or discontinuous ion-exchange chromatography for fractionation of CPK isoenzymes. The ion-exchange chromatography was accomplished on a DEAE-Sephadex A-50 ion-exchange resin filled in a chromatographic column utilizing a sample addition of blood sera followed by elution using a series of two or three elutants. In the Mercer procedure, buffered aqueous solutions of 0.1 molar and 0.2 molar sodium chloride, each with a pH of 8, were respectively used and, in some cases, a third elutant involving a buffered aqueous solution of 0.3 molar sodium chloride having a pH of 7.0 was employed. In the fractionation, the initial sample, generally 1 ml, was fractionated with multiple single 1 ml eluate fractions being collected. Each of these fractions was then assayed for CPK activity. This discontinuous ion-exchange chromatographic fractionation of CPK isoenzymes results in the production of some ten separate fractions, each of which has to be analyzed and assayed for CPK activity. The end results achieved in this discontinuous ion-exchange chromatographic procedure is a plot of assays of CPK activity for each of the fractions against fraction numbers. Again, as was the situation with the gradient technique of Takahashi et al, supra, analysis of a large number of fractions is required to use this method for CPK isoenzyme analysis.
Further, due to the difficulties in electrophoretic analysis, investigators such as L. E. Nathan et al, Clin. Chem., 19, 1036 (1973) have evaluated and described techniques for ion-exchange chromatography on DEAE-Sephadex of LDH enzymes to obtain LDH.sub.5 for assay as a diagnostic tool in hepatic disorders.
On examination of the prior art, of which the above is believed to be respresentative, it can be seen that while ion-exchange chromatographic separation is a definite advance over electrophoretic procedures, problems still exist relative to the prior art methods of separation and isolation of the clinically important CPK isoenzymes and LDH isoenzymes by ion-exchange chromatography in that, in these prior art methods, separation has been of the individual isoenzymes, generally involving a large number of samples which must be subsequently assayed for determination of the CPK and LDH isoenzyme levels, and, due to the inherent problems in the procedures described in the prior art, has resulted in the collection of fractions of various isoenzymes which need not be separated and collected for clinical evaluation, in particular of myocardial disorders. In addition, the procedures described in the prior art relative to the use of ion-exchange chromatography to separate CPK or LDH enzymes have resulted in, not only the production of multiple fractions, but have also resulted in excessive dilution of the eluates due to the nature of the sequential elution involved in the described procedures.